The overall goal of this project is to characterize the molecular mechanisms of olfactory signal transduction. The olfactory receptor neuron (ORN) is a unique CNS model system. It is capable of regeneration from a maintained population of basal cells. Although it is structurally adapted to perform its chemosensory function at the interface of the environment and the CNS, the ORN utilizes signal transduction proteins found elsewhere in the CNS, and therefore its complex mechanisms for signal transduction should provide insight into transduction elsewhere in the CNS. Two experimental paradigms have been develop for these studies. We have developed a method for primary culture of rat ORN's. To permit direct assay of enzymes such as adenylyl cyclase (AC) and phosphodiesterase (PDE) in relatively intact cells, we have developed a method for primary culture of rat ORN's. To permit direct assay of enzymes such as adenylyl cyclase (AC) and phosphodiesterase (PDE) in relatively intact cells, we have developed a method for a-toxin permeabilization of cells while still in monolayer culture. A second paradigm utilizes rat olfactory cilia isolated by calcium (Ca2) shock in a rapid stop-flow apparatus to assay second messenger responses at rapid time points. The first specific aim will be to characterize the regulation of the cAMP in these two model systems. Two parameters, the free Ca2+ concentration and the effect of phosphorylation, will be determined. The relative contribution of AC activation and PDE activation to the resulting cAMP signal will be determined in isolated cilia a number of specific PDE inhibitors will be utilized. AC and PDE activities can be directly measured in olfactory cultures treated with a-toxin. The second specific aim will investigate the role of the phosphoinositide (PI) system in olfaction. Using the rapid stopflow device to assay changes in InsP in rat cilia, we will determine the generality of this response and its regulation by Ca2+. The ability of odorant or Ca2+ to affect InsP binding to its receptor will also be determined. Using [H]-myoinositol to metabolically primary cultures or a radioassay to detect InsP levels, we will evaluate the ability of a number of odorant to stimulate PI turnover. We will examine the desensitization and resensitization of this response. Subsequent experiments will address the ability of protein kinases and of Ca2+ to modulate this response, thereby clarifying the mechanisms of desensitization. The systems we have adapted represent complementary approaches and present opportunities to study biochemical aspects of olfactory signal transduction, with implications for transduction and desensitization and other G-protein-mediated receptor systems.